Preparation of crystalline dihydrostreptomycin hydrochloride



Patented Apr. 22, 1952 PREPARATION OF CRYSTALLINE DIHYDRO- STREPTOMYCINHYDROCHLORIDE Frank J. Wolf, Westfield, N. J assignor to Merck & 00.,Inc., Bahway, N. J a corporation of New Jersey No Drawing. ApplicationMay 19, 1950, Serial No. 163,105

3 Claims. (01. 260-210 This application is a continuation-in-part of mypending application Serial No. 31,200, filed June 4, 1948, nowabandoned.

This invention relates to the preparation of improved antibioticsubstances and more particularly to the preparation ofdihydrostreptomycin in pure crystalline form.

Dihydrostreptomycin has been prepared from streptomycin by catalyticreduction in water solution in accordance with the procedure in apublication by Peck, I-Iofihine and Folkers in J. A. C. S. 68, 1390(1946). Dihydrostreptomycin can also be prepared from the streptomycinhydrochloride-calcium chloride complex by catalytic reduction thereof inaqueous methanol solution. By either of these procedures, however, thedihydrostreptomycin has been recovered only in the amorphous form andeven upon purification by conventional procedures has not been obtainedcompletely pure or even within the range of 5% of absolute purity. Theneed has nevertheless been recognized for obtaining dihydrostreptomycinin pure form to provide an accurate standard for purity determinationsand for clinical evaluation of the compound itself.

Over an extended period of time many at- In preparing crystallinedihydrostreptomycin tempts were made to purify dihydrostreptomycin bycrystallization from various solvents and solvent mixtures. So manyattempts of this sort had been made without obtaining any crystallinedihydrostreptomycin that it appeared quite likely that this product wasone which could not be obtained in crystalline form. The distinctpractical advantage of obtaining a crystalline product, however, spurredfurther investigations in spite of these negative indications.

As a result of this further investigation it has now been discoveredthat by following a particular procedure it is possible to produce smallcrystals of dihydrostreptomycin and that by using these crystals asseeds it is possible to prepare crystalline dihydrostreptomycin in largequantities.

The first crystal of dihydrostreptomycin hydrochloride was obtained whena methanol solution of dihydrostreptomycin hydrochloride (40 mg./cc.)was added to about 15 volumes of acetone and allowed to stand. Insteadof the usual formation of a fiocculent precipitate the solution merelyremained turbid. Upon allowing this turbid solution to stand overnightit was found that a small crystal had formed. This crystal was brokenand used to seed a number of other solutions of dihydrostreptomycinhydrochloride in methanol made turbid with acetone, and a substantialamount of crystalline dihydrostreptomycin hydrochloride was thusobtained.

the amorphous material in as pure a .form as possible. This can beaccomplished in various ways. For example, dihydrostreptomycinhydrochloride can be converted to the crystalline.

helianthate salt which is then purified by recrystallization and thenreacting same with hydrochloric acid to recover purified amorphousdihydrostreptomycin hydrochloride. On the other hand, if thedihydrostreptomycin hydrochloride is'prepared by reduction ofstreptomycin hydrochloride which has previously been purified bypreparation of the helianthate or streptomycincalcium chloride doublesalt, further purification of the dihydrostreptomycin hydrochloride isnot necessary.

For the crystallization of dihydrostreptomycin in accordance with thepresent invention an es-, sentially anhydrous solution of purifieddihydrostreptomycin hydrochloride in methanol is prepared. Upon seedingwithcrystals of the compound and stirring, crystallinedihydrostreptomycin hydrochloride is obtained. The rate ofcrystallization is materially speeded up by decreasing the relativeamount of methanol in said solution so that a condition ofsupersaturation is obtained. This can be achieved in a number of Waysas, for example, by adding additional dihydrostreptomycin hydrochlorideor evaporating part of the methanol until the amount ofdihydrostreptomycin hydrochloride ranges from 300 to 500 mg/ml.

When treating more dilute solutions, however, the supersaturation can beobtained by adding a solvent miscible with methanolin whichdihydrostreptomycin hydrochloride is relatively insoluble. A largenumber of solvents are suitable for this purpose, the most satisfactorybeing acetone, isopropanol and ethanol. The amount of added solventwill, of course, depend upon the particular solvent selected and thestarting concentration of dihydrostreptomycin in methanol. It is found,however, that when the proper dedegree of supersaturation is produced inthe crystallization mixture, as evidenced by the appearance of distinctturbidity in the mixture, a yield'of about of crystalline product isread ily obtained.

In order to foster maximum crystal formation the crystallization mixtureafter seeding is allowed to said with frequent shaking or agitation fora period of from about 15 to 40 hours. It should also be noted that byconcentrating the mother liquors remaining after the removal of thefirst crop of crystals and proceeding as before, additional quantitiesofcrystalline product ployed in drying the crystals. When dried undervacuum for periods up to 24 hours at temperatures ranging from roomtemperature to about 60 C. the crystals contain bound methanol and havea. distinct crystal form when subjected to X-ray and microscopicexamination. On the other hand, if the crystals are further dried byheating under vacuum for about 4 hours at a temperature of about 100 C.crystals are obtained which are essentially free of methanol and have adifferent characteristic crystal form.

The amount of methanol in the methanol containing crystals varies withthe drying temperature. Heating under vacuum at a temperature of about56 C. gives a product containing (by analysis) 3 to 4% of methanol whichis quite firmly bound, indicating that the amount of firm- 1y boundmethanol is one mole per mole of dihydrostreptomycin hydrochloride.Heating at room temperature, however, gives crystals which (by analysis)contain about 10-11% of methanol, indicating that this product containsabout three molar equivalents of methanol, 1. e., one mole being thefirmly bound methanol and two moles being loosely bound methanol not inthe crystalline structure. This would account for the fact that crystalsdried at room temperature and at 56 C., although differing in methanolcontent, have the same crystal form.

The following examples show how the process of the present invention canbe carried out, but it is to be understood that these examples are givenby way of illustration and not of limitation.

EXAMPLE 1 Streptomycin-calcium chloride complex (100 gm.) was dissolvedin 500 cc. of water and the solution shaken with hydrogen in thepresence of 1 gm. of platinum oxide. After absorption of hydrogen hadceased the solution was filtered from the catalyst and poured withstirring into 10 l. of acetone containing about 100 gm. of Supercell (adiatomaceous earth filter aid). The Supercel slurry was filtered, washedwith acetone, and the dihydrostreptomycin removed by leaching the cakewith two 200 cc. portions of methanol and finally washing with 100 cc.of methanol. Amorphous dihydrostreptomycin hydrochloride, essentiallyfree from calcium chloride, was obtained by pouring the methanolsolution into 5 l. of isopropanol. After drying, '84 gm. of theamorphous product was obtained.

For crystallization the amorphous product was dissolved in 300 cc. ofmethanol, seeded and stirred. After 40, hours stirring the crystallineproduct was filtered and washed with 100 cc. portions of methanol, 80-20methanol-acetone, 50-50 methanol-acetone, and acetone and dried,yielding 64 gm. (76% yield) of crystalline prodnot. In a duplicate runfollowing the same procedure a yield of 66 gm. of crystalline product(83%) and gm. of amorphous material was obtained.

The crystalline products were recrystallized by dissolving 5 gm. in '7cc. of boiling methanol, adding acetone to the point of turbidity (45-50cc.) seeding and stirring. Yields of 3.3 and 3.7 gm. (66 and 74%respectively) were obtained. The products, dried at about 56 C. invacuo,

contain about 4% of methanol, have a specific rotation of 91 to --94 andcontain no ash.

" Crystalline dihydrostreptomycin hydrochloride, 56 gm. was dissolved inhot methanol (1400 cc.) and the resultant slightly turbid solutionfiltered and diluted with acetone to the point of turbidity (ca. 1400cc.) seeded and stirred for about 40 hours. The product was filtered,washed, and dried yielding 49.0 gm. (80.5%) of crystalline first crop.The mother liquor and washes were combined and concentrated to about 300cc. acetone again added and seeded. A crystalline product amounting to8.2 gm. (13.5%) was obtained. The crystals when dried in vacuo at about56 C. contained about 4% of methanol, indicating the presence of onemole of methanol per mole of dihydrostreptomycin hydrochloride in thecrystal structure. Precipitation of the washes and mother liquors inacetone yielded 2 gm. of additional amorphous material.

EXAMPLE 3 A solution of 1800 gm. of crystalline streptomycin-calciumchloride double salt equivalent to 1600 gm. of dry product was dissolvedby vigorous stirring in 600 cc. of water and 3 l. of methanol. Theviscous solution was reduced using 1.8 gm. of platinum oxide catalyst.After the reduction was complete the mixture was filtered from thecatalyst and distilled under reduced pressure with continuous additionof methanol to the distillation vessel. After about 15 l. of methanolhad been distilled from the mixture, analysis for water indicated thepresence of about 200 gm. of water. Some suspended colloidal catalystnot removed by the initial filtration was removed by adding 10 gm. ofactivated carbon and filtering. The filtrate, volume about 5 1., wasdiluted with 1 l. of acetone. The mixture was seeded with 10 gm. ofcrystalline dihydrostreptomycin hydrochloride and stirred for 16 hours.The product was filtered, washed with l l. of methanol, then 50-50methanol-acetone and finally with acetone and dried. The dry product(770 gm.; 60% yield) contained 0.2% sulfated ash. Upon concentration andseeding of the mother liquor a second crop of 40 gm. was obtained.

EXAMPLE 4 A solution of 5 gm. of crystalline dihydrostreptomycinhydrochloride in '7 5 cc. of hot methanol was diluted with isopropanoluntil a faint turbidity remained in the solution (3.8 cc. wererequired). The mixture was seeded with crystal line dihydrostreptomycinhydrochloride and allowed to stand 72 hours with occasional shaking. Theproduct was filtered, washed with methanol-isopropanol mixture thenisopropanol and dried, in vacuo at about 56 C., yielding 2.9 gm. (68%)of crystalline product.

Pertinent data concerning this product are tabulated below and comparedwith properties of amorphous dihydrostreptomycin hydrochloride.

Properties Amorphous Crystalline Potency (streptomycin units per mg, 500600.

B. Subtilis cup assay). 7 Toxicity LD5O (mg/20 g. mouse) 3.84.2...4.44.7. Rotation a so. (0:1 in water). -87-88 92-94. Methanol solubilityMiscible. 1 gm. in 15 cc. Crystalline Oharacteris None... Purity I 97%or greater.

1 Crystalline characteristics.

X-ray diffraction pattern spacings and relative intensities measured bythe Norelco Geiger-counter X-ray spectrometer are tabulated below for asample of dihydrostreptomycin trihydrochloride, dried for 4 hours at 100C.

Relative Spacing, A Intensity,

Percent Other crystalline characteristics are as follows:

1 For the product as dried at 56 C.

EXAMPLE 5 Samples of crystalline dihydrostreptomycin hydrochloride wereprepared from various solvents and concentrations. The product wascentrifuged and the damp solid examined by X-ray. In addition one of thesamples was air-dried at room temperature, dried in vacuo at roomtemperature, 56 C. and 100 C. With the exception of the 100 C. driedmaterial, all samples had the same crystalline form when examined byX-ray although some samples appeared different to the eye.

M ethod of crystallization v SAMPLE 1 1A methanol solution ofdihydrostreptomycin hydrochloride (200 mg./cc.) was diluted with volumeof isopropanol. Slow crystallization (seeded) on standing produced largerectangular crystals.

' SAMPLE 2 A methanol solution of dihydrostreptomycin hydrochloride (400mg./cc.) and 32 mg./cc. of calcium chloride. Slowly crystallized(seeded). Crystals formed crust on bottom of the flask.

SAMPLE. 3 jMethanol solution of dihydrostreptomycin hydrfochloride (200mg./cc.). A slight amount of seed crystals was added and the crystalsformed slowly.

SAMPLE 4 A methanol solution of dihydrostreptomycin hydrochloride (400mg./cc.) was seeded and stirred forming fine crystals.

SAMPLE 5 A methanol solution of dihydrostreptomycin hydrochloride (200men/cc.) and calcium chloride (80 mg./cc.) seeded was allowed to standforming large coarse crystals. This sample was airdried. Methanolcontent%.

Vacuum dried-room temperature-11% methanol Vacuum dried-56 C'.--4%methanol Yacuum dried-400 C.0.4% methanol Upon X-ray and microscopicexamination, all of the undried samples and the samples dried in vacuoat 56 C. or below were found to have the same crystal form, while thesample dried at 100 C. had a different and distinct crystal form.

7 Comparative data for samples of crystalline dihydrostreptomycinhydrochloride dried under different conditions are tabulated below.

Crystals Dried in vacuo Characteristic Sample A Sample B Sample 0 (atC.) (at 56 C.) (at 100 C.)

a l 528i. 002 l. 532:1; 002 1.5501002 Index of refraction 5 l 544:!:.002 l. 5485:. 002 1. 56611:. 002 'y l 5663:. 002 1. 5663:. 002 1. 5785:.002 Birefringence +0. 006 -0. 002 0. 004 Axial Angle 2V (calculated),de-

grees 80 80 7s Methanol Content, percent l0. 2 3.1 0, 4 X-rayDiffraction Pattern Crystalline (orm 3 A a A B 56tXdray DiffractionPattern for Crystals Dried at 25- Relative Relative Spacing, AIntensity, Spacing, A Intensity, Per Cent Per Cent 1 X-ray DiffractionPattern for Crystals Dried at C. 3 The crystals form designations A andB are arbitrary designations given to the two types of crystals asidentified by the characteristic indices of refraction and Xraydiffraction patterns.

Relative Spacing, l Intensity, Per Cent The remaining characteristicssuch as extinction, sign of elongation, pleochroism, and crystal systemare the same for both crystal ,forms.

It has been found that after a number of crystallization ofdihydrostreptomycin hydrochloride have been carried out in a particularenvironment, crystallization of additional amounts ofdihydrostreptomycin hydrochloride can some.- times be efiected withoutthe actual addition of seed crystals. This is probably due to thepresence of minute crystals of diihydrostreptomycin hydrochloride in theapparatus or atmosphere of the environment. In order to provide optimumcontrol of the crystallization and to efiect crystallization in theshortest possible time, it is preferable, however, to add seed crystalsto the supersaturated solution rather than to rely on the seeding actionof minute crystals in the environment. I

It is to be understood that novelty of the herein described processresides not only in the procedures for crystallizing dihydrostreptomycinhydrochloride but also in the preferred adaptation of the processwherein the streptomycin hydrochloride calcium chloride complex salt iscatalytically reduced and the dihydrostreptomycin hydrochloride thusobtained in amorphous form 'is converted to the crystalline product bythe procedures herein described.

In this preferred adaptation of the process the manner of removingcalcium chloride from the reaction mixturefoll-owing the reduction is ofprimary significance. This step involves precipitation ofdihydrostreptomycin hydrochloride from the methanolic reduction mixturecontaining calcium chloride with isopropanol, thus forming an amorphousprecipitate of 'dihydrostreptomycin hydrochloride which is readilyrecovered by filtration. This calcium chloride-free material is wellsuited for use in the preparation of crystalline dihydrostreptomycin andmay also be utilized as a superior quality of amorphous product.

It should further be understood that throughout the specification andclaims reference to dihydrostreptomycin hydrochloride denotes thecompound which contains 3 molecules of HCl, i. e., dihydrostreptomycintrihydrochloride.

Various changes and modifications in the foregoing procedures will occurto those versed in the art, and to the extent that such changes andmodifications fall within the purview of the appended claims, it is tobe understood that they constitute part of my invention.

I claim:

1. The process for preparing crystalline dihydrostrept-omycinhydrochloride that comprises preparing a methanol solution ordihydrostreptomycin hydrochloride of about 40 rug/cc. concentration,adding a volume of this solution to about .15 volumes of acetone, andallowing the resulting turbid solution to stand until crystals 7 areformed, thereby obtaining crystalline dihydrostreptomycin hydrochloridecontaining methanol of crystallization.

2. The process for preparing crystalline dihydrostreptomycinhydrochloride that comprises preparing a methanol solution ofdihydrostrep-' tomycin hydrochloride of about 40 mg./cc. concentration,adding a volume of this solution to about '15 volumes oi acetone, andallowing the resulting turbid solution to stand until crystals areformed, thereby obtaining crystalline dihydrostreptomycin hydrochloridecontaining methanol of-crystallization, and heating the same undervacuum at a temperature of about 100 C. thereby obtaining methanol-freecrystalline dihydrostreptomycin hydrochloride.

3. The process for preparing crystalline dihydrostreptomycinhydrochloride that comprises preparing a methanol solution ofdihydrostreptomycin hydrochloride of about 40 mg./cc. concentration,adding a volume of this solution to about 15 volumes of acetone, andallowing the resulting turbid solution to stand until crystals areformed, thereby obtaining crystalline dihydrostreptomycin hydrochloridecontaining methanol of crystallization, and utilizing the crystals thereobtained to seed a supersaturated methanolic solution ofdihydrostreptomycin hydrochloride in the production of additionalcrystalline dihydostreptomycin hydrochloride.

FRANK J. WOLF.

REFERENCES CITED The following references are of record in the file ofthis patent:

UNITED STATES PATENTS Number Name Date 7 2,446,102 Peck July 27, 19482,462,175 Folkers Feb. 22, 1949 2,498,574 Peck Feb. 21, 1950 2,552,547 7Fried et a1 May 15, 1951 OTHER REFERENCES Le Page, J. Biol. Chem., vol.162 (1946), page 167.

Fried et al., Jour. Amer. Chem. Soc, vol. 69, page 83 (1947).

Bartz et al., Jour. Amer. Chem. Soc, vol. 68,

.pages 2163-2166 (1946) I. A. Solomons et al., Science, vol. 109, page

1. THE PROCESS FOR PREPARING CRYSTALLINE DIHYDROSTREPTOMYCINHYDROCHLORIDE THAT COMPRISES PREPARING A METHANOL SOLUTION OFDIHYDROSTREPTOMYCIN HYDROCHLORIDE OF ABOUT 40 MG./CC. CONCENTRATION,ADDING A VOLUME OF THIS SOLUTION TO ABOUT 15 VOLUMES OF ACETONE, ANDALLOWING THE RESULTING TURBID SOLUTION TO STAND UNTIL CRYSTALS AREFORMED, THEREBY OBTAINING CRYSTALLINE DIHYDROSTREPTOMYCIN HYDROCHLORIDECONTAINING METHANOL OF CRYSTALLIZATION.